Testing intra-species variation in allocation to growth and defense in rubber tree (Hevea brasiliensis).

Background: Plants allocate resources to growth, defense, and stress resistance, and resource availability can affect the balance between these allocations. Allocation patterns are well-known to differ among species, but what controls possible intra-specific trade-offs and if variation in growth vs. defense potentially evolves in adaptation to resource availability. Methods: We measured growth and defense in a provenance trial of rubber trees (Hevea brasiliensis) with clones originating from the Amazon basin. To test hypotheses on the allocation to growth vs. defense, we relate biomass growth and latex production to wood and leaf traits, to climate and soil variables from the location of origin, and to the genetic relatedness of the Hevea clones. Results: Contrary to expectations, there was no trade-off between growth and defense, but latex yield and biomass growth were positively correlated, and both increased with tree size. The absence of a trade-off may be attributed to the high resource availability in a plantation, allowing trees to allocate resources to both growth and defense. Growth was weakly correlated with leaf traits, such as leaf mass per area, intrinsic water use efficiency, and leaf nitrogen content, but the relative investment in growth vs. defense was not associated with specific traits or environmental variables. Wood and leaf traits showed clinal correlations to the rainfall and soil variables of the places of origin. These traits exhibited strong phylogenetic signals, highlighting the role of genetic factors in trait variation and adaptation. The study provides insights into the interplay between resource allocation, environmental adaptations, and genetic factors in trees. However, the underlying drivers for the high variation of latex production in one of the commercially most important tree species remains unexplained.

A transcriptome-based association study of growth, wood quality, and oleoresin traits in a slash pine breeding population.

Slash pine (Pinus elliottii Engelm.) is an important timber and resin species in the United States, China, Brazil and other countries. Understanding the genetic basis of these traits will accelerate its breeding progress. We carried out a genome-wide association study (GWAS), transcriptome-wide association study (TWAS) and weighted gene co-expression network analysis (WGCNA) for growth, wood quality, and oleoresin traits using 240 unrelated individuals from a Chinese slash pine breeding population. We developed high quality 53,229 single nucleotide polymorphisms (SNPs). Our analysis reveals three main results: (1) the Chinese breeding population can be divided into three genetic groups with a mean inbreeding coefficient of 0.137; (2) 32 SNPs significantly were associated with growth and oleoresin traits, accounting for the phenotypic variance ranging from 12.3% to 21.8% and from 10.6% to 16.7%, respectively; and (3) six genes encoding PeTLP, PeAP2/ERF, PePUP9, PeSLP, PeHSP, and PeOCT1 proteins were identified and validated by quantitative real time polymerase chain reaction for their association with growth and oleoresin traits. These results could be useful for tree breeding and functional studies in advanced slash pine breeding program.

DNA from wood - A simple approach facing a challenging matrix - A preliminary study.

Plant DNA barcoding has proven to be a handy tool for identifying botanical species. However, extracting DNA from woody materials is often a challenging task. Forensic applications, therefore, must be able to overcome the technical difficulties of this nature. A simple and successful adaptation, through a widely used method in forensic laboratories, using chemistry based on magnetic DNA isolation technology and a robotic platform, is presented here. The model case was the application of this adapted DNA extraction method for the identification of Aspidosperma spp., a genus comprising several species in all biomes of Brazil, including Cerrado and Rain Forest. Such technology adaptation can aid in the identification of seized material and help in investigations involving illegal logging and deforestation, ultimately contributing to environmental protection.

Analysis of NAC Domain Transcription Factor Genes of Tectona grandis L.f. Involved in Secondary Cell Wall Deposition.

NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus, Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes (Tg11g04450-VND2 and Tg15g08390-VND4) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches.

Quantitative genetic parameters for growth and wood properties in Eucalyptus "urograndis" hybrid using near-infrared phenotyping and genome-wide SNP-based relationships.

A thorough understanding of the heritability, genetic correlations and additive and non-additive variance components of tree growth and wood properties is a requisite for effective tree breeding. This knowledge is essential to maximize genetic gain, that is, the amount of increase in trait performance achieved annually through directional selection. Understanding the genetic attributes of traits targeted by breeding is also important to sustain decade-long genetic progress, that is, the progress made by increasing the average genetic value of the offspring as compared to that of the parental generation. In this study, we report quantitative genetic parameters for fifteen growth, wood chemical and physical traits for the world-famous Eucalyptus urograndis hybrid (E. grandis × E. urophylla). These traits directly impact the optimal use of wood for cellulose pulp, paper, and energy production. A population of 1,000 trees sampled in a progeny trial was phenotyped directly or following the development and use of near-infrared spectroscopy calibration models. Trees were genotyped with 33,398 SNPs and 24,001 DArT-seq genome-wide markers and genomic realized relationship matrices (GRM) were used for parameter estimation with an individual-tree additive-dominant mixed model. Wood chemical properties and wood density showed stronger genetic control than growth, cellulose and fiber traits. Additive effects are the main drivers of genetic variation for all traits, but dominance plays an equally or more important role for growth, singularly in this hybrid. GRM´s with >10,000 markers provided stable relationships estimates and more accurate parameters than pedigrees by capturing the full genetic relationships among individuals and disentangling the non-additive from the additive genetic component. Low correlations between growth and wood properties indicate that simultaneous selection for wood traits can be applied with minor effects on genetic gain for growth. Conversely, moderate to strong correlations between wood density and chemical traits exist, likely due to their interdependency on cell wall structure such that responses to selection will be connected for these traits. Our results illustrate the advantage of using genome-wide marker data to inform tree breeding in general and have important consequences for operational breeding of eucalypt urograndis hybrids.

Improving genomic prediction of growth and wood traits in Eucalyptus using phenotypes from non-genotyped trees by single-step GBLUP.

Genomic Best Linear Unbiased Prediction (GBLUP) in tree breeding typically only uses information from genotyped trees. However, information from phenotyped but non-genotyped trees can also be highly valuable. The single-step GBLUP approach (ssGBLUP) allows genomic prediction to take into account both genotyped and non-genotyped trees simultaneously in a single evaluation. In this study, we investigated the advantage, in terms of breeding value accuracy and bias, of including phenotypic observation from non-genotyped trees in a standard tree GBLUP evaluation. We compared the efficiency of the conventional pedigree-based (ABLUP), GBLUP and ssGBLUP approaches to evaluate eight growth and wood quality traits in a Eucalyptus hybrid population, genotyped with 33,398 single nucleotide polymorphisms (SNPs) using the EucHIP60k. Theoretical accuracies, predictive ability and bias were calculated by ten-fold cross validation on all traits. The use of additional phenotypic information from non-genotyped trees by means of ssGBLUP provided higher predictive ability (from 37% to 75%) and lower prediction bias (from 21% to 73%) for the genetic component of non-phenotyped but genotyped trees when compared to GBLUP. The increase (decrease) in the prediction accuracy (bias) became stronger as trait heritability decreased. We concluded that ssGBLUP is a promising breeding tool to improve accuracies and bias over classical GBLUP for genomic evaluation in Eucalyptus breeding practice.

Regional heritability mapping and genome-wide association identify loci for complex growth, wood and disease resistance traits in Eucalyptus.

Although genome-wide association studies (GWAS) have provided valuable insights into the decoding of the relationships between sequence variation and complex phenotypes, they have explained little heritability. Regional heritability mapping (RHM) provides heritability estimates for genomic segments containing both common and rare allelic effects that individually contribute too little variance to be detected by GWAS. We carried out GWAS and RHM for seven growth, wood and disease resistance traits in a breeding population of 768 Eucalyptus hybrid trees using EuCHIP60K. Total genomic heritabilities accounted for large proportions (64-89%) of pedigree-based trait heritabilities, providing additional evidence that complex traits in eucalypts are controlled by many sequence variants across the frequency spectrum, each with small contributions to the phenotypic variance. RHM detected 26 quantitative trait loci (QTLs) encompassing 2191 single nucleotide polymorphisms (SNPs), whereas GWAS detected 13 single SNP-trait associations. RHM and GWAS QTLs individually explained 5-15% and 4-6% of the genomic heritability, respectively. RHM was superior to GWAS in capturing larger proportions of genomic heritability. Equated to previously mapped QTLs, our results highlighted genomic regions for further examination towards gene discovery. RHM-QTLs bearing a combination of common and rare variants could be useful enhancements to incorporate prior knowledge of the underlying genetic architecture in genomic prediction models.

Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp.

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 µg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.

Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in eucalyptus globulus

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus...

Genomic selection for growth and wood quality in Eucalyptus: capturing the missing heritability and accelerating breeding for complex traits in forest trees.

• Genomic selection (GS) is expected to cause a paradigm shift in tree breeding by improving its speed and efficiency. By fitting all the genome-wide markers concurrently, GS can capture most of the 'missing heritability' of complex traits that quantitative trait locus (QTL) and association mapping classically fail to explain. Experimental support of GS is now required. • The effectiveness of GS was assessed in two unrelated Eucalyptus breeding populations with contrasting effective population sizes (N(e) = 11 and 51) genotyped with > 3000 DArT markers. Prediction models were developed for tree circumference and height growth, wood specific gravity and pulp yield using random regression best linear unbiased predictor (BLUP). • Accuracies of GS varied between 0.55 and 0.88, matching the accuracies achieved by conventional phenotypic selection. Substantial proportions (74-97%) of trait heritability were captured by fitting all genome-wide markers simultaneously. Genomic regions explaining trait variation largely coincided between populations, although GS models predicted poorly across populations, likely as a result of variable patterns of linkage disequilibrium, inconsistent allelic effects and genotype × environment interaction. • GS brings a new perspective to the understanding of quantitative trait variation in forest trees and provides a revolutionary tool for applied tree improvement. Nevertheless population-specific predictive models will likely drive the initial applications of GS in forest tree breeding.